Most human cancers are characterized by chromosome number alterations. Molecular defects that initiate or promote losses or gains of whole chromosomes remain largely unknown. In some cancers with chromosomal instability, mutations have been identified in genes that encode for mitotic checkpoint proteins, such as Bub1 BubR1 and Mad2. These proteins are parts of an intricate molecular machine that blocks the onset of anaphase until all chromosomes are properly attached to the mitotic spindle apparatus and aligned at the metaphase plate. Following disassembly of the nuclear envelope at mitosis, the nucleocytoplasmic transport factors Ran and importin alpha/beta become key regulators of mitotic spindle assembly. Nup98, another nuclear transport factor, is a frequent target of genetic alterations in acute leukemias. Nup98 contains a motif, termed GLEBS, that directs binding to the putative mRNA export factor Rae1. We have recently shown that a GLEBS motif is present in the mitotic checkpoint proteins Bub1 and BubR1, where it serves as a binding site for the Rae1-related mitotic checkpoint protein Bub3. Using a gene-knockout approach, we have determined that Nup98 and Rae1 not only share sequence homology with Bub proteins, but are required for mitotic checkpoint function as well. The overall goal of this proposal is to define the role of Nup98 and its binding partner Rae1 in the maintenance of chromosomal stability and tumor formation. In the first aim, we will determine the mechanism by which Nup98 regulates mitotic checkpoint function and chromosomal stability. In the second aim, we will examine the role of Nup98 in normal and neoplastic growth by tissue-specific disruption of Nup98 in mice. In the third aim, we will focus on the role of Rae1 in mitotic checkpoint function, maintenance of chromosomal stability, and tumor formation. Information gained from these studies will provide a framework for understanding the mechanisms by which nuclear transport factors contribute to genetic instability and cancer.